Mechanisms of ligand binding by monoclonal anti-fluorescyl antibodies.
Binding of fluorescyl ligand by five IgG anti-fluorescyl hybridoma proteins (4-4-20, 6-10-6, 20-4-4, 20-19-=1, 20-20-3) was examined. Relative reduction in fluorescence of bound fluorescein, deuterium oxide (D2O)-induced enhancement of fluorescence, and the effects of pH on binding kinetics were measured for each clone. Individual hybridoma proteins (all of which bind fluorescein with relatively high affinity) exhibited significant differences in the relative contribution of various forces (hydrophobicity, hydrogen bonding, ionic interactions) to binding and hence, affinity. The extent of such variations in binding mechanisms among monoclonal antibodies binding the same hapten is indicative of the extreme functional diversity of active sites. In addition, ligand binding by clone 20-20-3 was examined in greater detail. ABsorption spectra of ligand bound by purified intact antibody, Fab fragments, and reassociated heavy and light chains indicated that protonation of the fluorescyl ligand by a residue within the active site contributed significantly to the binding free energy. Comparative dissociation rates of fluorescein and a structural analog, rhodamine 110, were used to quantitatively substantiate the contribution of this interaction. Association and dissociation rate studies with fluorescein and antibody indicated that: 1) the active site appeared to undergo a conformational change upon ligand binding, and 2) neither intact disulfides nor intersite cooperativity affected the dissociation rate of bound ligand. Observed mechanisms of ligand binding are discussed in terms of proposed mechanisms of antibody affinity maturation and diversity.
D000911:Antibodies, Monoclonal; D000936:Antigen-Antibody Complex; D005452:Fluoresceins; D006825:Hybridomas / Q000276:immunology; D006863:Hydrogen-Ion Concentration; D007074:Immunoglobulin G; D007143:Immunoglobulin Heavy Chains / Q000302:isolation & purification; D007147:Immunoglobulin Light Chains / Q000302:isolation & purification; D007700:Kinetics; D008433:Mathematics; D013050:Spectrometry, Fluorescence
D000911:Antibodies, Monoclonal; D000936:Antigen-Antibody Complex; D005452:Fluoresceins; D007074:Immunoglobulin G; D007143:Immunoglobulin Heavy Chains; D007147:Immunoglobulin Light Chains; C030864:5-aminofluorescein